Name: using real-time cell metabolic flux analyzer to monitor osteoblast bioenergetics
Uploaded: 2022-03-01T12:45:00
Description: Watch this Scientific Journal Video about Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics at JoVE.com

This work was supported by the National Institute of Health (NIH) National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) Grant AR072123 and National Institute on Aging (NIA) Grant AG069795 (to ERR).

All the procedures were based on the guidelines and approval of the Institutional Animal Care and Use Committee at Vanderbilt University Medical center.
1. Preparation of reagents and assay setup
<ol>
	<li>Isolation and culturing of bone marrow stromal cells (also see the previous article30).
	<ol>
		<li>Prepare complete alpha minimum essential media (&#945;MEM) cell culture media by supplementing minimum essential media with alpha modification with 1...

<ol>
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B., Nichols, N., Nichols, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+studies+of+bone+in+vitro:+II.+The+metabolic+patterns+of+accretion+and+resorption.">Metabolic studies of bone in vitro: II. The metabolic patterns of accretion and resorption.</a> Journal of Biological Chemistry. 235, 1211-1214 (1960).</li><li>Adamek, G., Felix, R., Guenther, H. L., Fleisch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+oxidation+in+bone+tissue+and+bone+cells+in+culture.+Characterization+and+hormonal+influences.">Fatty acid oxidation in bone tissue and bone cells in culture. Characterization and hormonal influences.</a> The Biochemical Journal. 248 (1), 129-137 (1987).</li><li>Frey, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt-Lrp5+signaling+regulates+fatty+acid+metabolism+in+the+osteoblast.">Wnt-Lrp5 signaling regulates fatty acid metabolism in the osteoblast.</a> Molecular and Cellular Biology. 35 (11), 1979-1991 (2015).</li><li>Romero, N., Rogers, G., Neilson, A., Dranka, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantifying cellular ATP production rate using agilent seahorse XF technology. , (2018).</li><li>vander Windt, G., Chang, C., Pearce, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+bioenergetics+in+T+cells+using+a+Seahorse+Extracellular+Flux+Analyzer.">Measuring bioenergetics in T cells using a Seahorse Extracellular Flux Analyzer.</a> Current Protocols in Immunology. 113, 1 (2016).</li><li>Traba, J., Miozzo, P., Akkaya, B., Pierce, S. K., Akkaya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+protocol+to+analyze+glycolysis+and+mitochondrial+respiration+in+lymphocytes.">An optimized protocol to analyze glycolysis and mitochondrial respiration in lymphocytes.</a> Journal of Visualized Experiments:JoVE. (117), e54918 (2016).</li><li>Noel, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+metabolic+assay+of+3-dimensional+spheroid+co-cultures+of+pancreatic+cancer+cells+and+fibroblasts.">Preparation and metabolic assay of 3-dimensional spheroid co-cultures of pancreatic cancer cells and fibroblasts.</a> Journal of Visualized Experiments:JoVE. (126), e56081 (2017).</li><li>Nicholls, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+profile+experiment+using+C2C12+myoblast+cells.">Bioenergetic profile experiment using C2C12 myoblast cells.</a> Journal of Visualized Experiments: JoVE. (46), e2511 (2010).</li><li>Sakamuri, S. S. V. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+respiratory+function+in+isolated+cardiac+mitochondria+using+Seahorse+XFe24+Analyzer:+applications+for+aging+research.">Measurement of respiratory function in isolated cardiac mitochondria using Seahorse XFe24 Analyzer: applications for aging research.</a> GeroScience. 40 (3), 347-356 (2018).</li><li>. What are the advantages of using Seahorse XF technology Available from: <a target="_blank" href="https://wwwagilent.com/en/support/cell-analysis/advantages-of-using-xf-tech">https://wwwagilent.com/en/support/cell-analysis/advantages-of-using-xf-tech</a> (2018)</li><li>Horan, M. P., Pichaud, N., Ballard, J. W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Quantifying+mitochondrial+dysfunction+in+complex+diseases+of+aging.">Review: Quantifying mitochondrial dysfunction in complex diseases of aging.</a> Journals of Gerontology - Series A Biological Sciences and Medical Sciences. 67 (10), 1022-1035 (2012).</li><li>. XF cell energy phenotype test Available from: <a target="_blank" href="https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-assay-kits-reagents-cell-assay-media/seahorse-xf-cell-energy-phenotype-test-kit-740884">https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-assay-kits-reagents-cell-assay-media/seahorse-xf-cell-energy-phenotype-test-kit-740884</a> (2021)</li><li>Leung, D. T. H., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+oxidative+stress:+Mitochondrial+function+using+the+seahorse+system.">Measurement of oxidative stress: Mitochondrial function using the seahorse system.</a> Methods in Molecular Biology. 1710, 285-293 (2018).</li><li>. XF ATP rate assay Available from: <a target="_blank" href="https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-assay-kits-reagents-cell-assay-media/seahorse-xf-cell-energy-phenotype-test-kit-740889">https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-assay-kits-reagents-cell-assay-media/seahorse-xf-cell-energy-phenotype-test-kit-740889</a> (2021)</li><li>. XF cell mito stress test Available from: <a target="_blank" href="https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-assay-kits-reagents-cell-assay-media/seahorse-xf-cell-energy-phenotype-test-kit-740885">https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-assay-kits-reagents-cell-assay-media/seahorse-xf-cell-energy-phenotype-test-kit-740885</a> (2021)</li><li>. XF cell mito fuel flex test Available from: <a target="_blank" href="https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-assay-kits-reagents-cell-assay-media/seahorse-xf-cell-energy-phenotype-test-kit-740888">https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-assay-kits-reagents-cell-assay-media/seahorse-xf-cell-energy-phenotype-test-kit-740888</a> (2021)</li><li>Maridas, D. E., Rendina-Ruedy, E., Le, P. T., Rosen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+differentiation+of+bone+marrow+stromal+cells+and+osteoclast+progenitors+from+mice.">Isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors from mice.</a> Journal of Visualized Experiments: JoVE. 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N., Jastroch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+interpretation+of+microplate-based+oxygen+consumption+and+pH+data.">Analysis and interpretation of microplate-based oxygen consumption and pH data.</a> Methods in Enzymology. 547, 309-354 (2014).</li><li>Kam, Y., Jastromb, N., Clayton, J., Held, P., Dranka, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normalization of agilent seahorse XF data by in-situ cell counting using a BioTek cytation 5 application note. , (2017).</li></ol>

The real-time cell metabolic flux analyzer can be used to explore cellular energetics under different conditions. The protocol illustrates the efficient isolation of BMSCs, culturing cells in appropriate cell culture plates, and their differentiation to mature osteoblasts, which can be used for various assays using the extracellular flux analyzer. Further, the critical steps of real-time cell metabolic flux assay, including hydration of sensor cartridges, loading of the injection ports, performing the assay, normalizatio...

The formation of bone by the osteoblast is accompanied by coordinated destruction or resorption of bones by osteoclasts. The balance between osteoblastic bone formation and osteoclast resorption is a coupled process describing bone turnover or remodeling, which is essential for skeletal homeostasis. Osteoblast dysfunction leads to impaired bone formation and results in various diseases, including osteoporosis1,2,3. Ex vivo/in vitro differentiation of bone marrow stromal stem cells (BMSCs) to osteoblast precursors and mature osteoblasts results in the formation and deposition of the mineralized bone matrix in the culture vessel over time4,5,6. This bone formation by the osteoblast requires a significant amount of cellular energy. Specifically, collagen synthesis and secretion have been shown to rely heavily on cellular ATP: ADP ratios, and presumably, mineralized vesicle trafficking and secretion require additional ATP7,8,9,10,11. Many researchers have demonstrated that the process of osteoblastogenesis and osteoblast function requires an adequate supply of energy to meet the metabolic demand of bone formation12,13,14,15,16. Therefore, the goal of this method is to characterize the bioenergetic status of primary, murine stromal cells throughout osteoblast differentiation using the real-time cell metabolic flux analyzer. These techniques aid in developing a better understanding of skeletal homeostasis, which may ultimately lead to the development of novel therapeutic options capable of improving skeletal disorders.
The real-time cell metabolic flux analyzer can be used to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live osteoblasts, which corresponds to mitochondrial and glycolytic ATP production, respectively. Fundamental to this methodology is the fact that one H+ ion per lactate is released during glycolysis in the conversion of glucose to lactate, which alters media pH reflected in the ECAR values. Conversely, during the TCA (tricarboxylic acid) cycle, oxidative phosphorylation via the mitochondria produces CO2 by utilizing or consuming oxygen, and therefore monitoring OCR is reflective of this metabolic process. The analyzer measures both OCR and ECAR in the extracellular microenvironment simultaneously and in real-time, which allows for tremendous potential when studying cellular bioenergetics6,17. Additionally, performing these assays is relatively straightforward and easily customizable depending on the experimental goal. Similar techniques have been employed to further understand T-cell metabolic regulation of the immune system18,19, cancer initiation, and progression20, along with multiple other cell types contributing to metabolic syndromes21,22.
The advantages of Real-time metabolic flux analyzer over alternative techniques include (1) the capability to measure cellular bioenergetics of live cells in real-time, (2) ability to perform assay with a relatively small number of cells (requires as low as 5,000 cells), (3) injection ports to parallelly manipulate multiple treatments in a high-throughput 96-well system, (4) use of radioactive label-free automated cell imager for normalization18,23,24. The following methods aim to provide a generalized but detailed description of monitoring cellular bioenergetics in murine BMSCs throughout osteoblast differentiation using the analyzer. It will include routinely performed assays; however, as with many techniques and methods, it is highly encouraged that individual labs determine specific details for their experiments.
Selection of assay and different types of assays available: A wide variety of assay kits and reagents are available to study the bioenergetics of cells while ensuring the reliability and consistency of the experimental results. Additionally, the desktop software also offers assay templates that can be easily customized. The assay can be defined based on the user&#39;s needs to measure different metabolic parameters. These assays can be modified in various ways based on the experimental goal and/or scientific question. For example, with four injection ports, multiple compounds can be injected into the assay media to analyze the cellular response specific to each metabolic pathway.
Cell energy phenotype test: This assay measures the live cells&#39; metabolic phenotype and metabolic potential. This assay is also recommended as the first step to get a generalized idea of pathway-specific metabolism. A mixture of oligomycin A-an inhibitor of ATP synthase and Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)-a mitochondrial uncoupling agent is injected to understand the cell energy potential. The injection of oligomycin A inhibits the synthesis of ATP, resulting in an increase in the rate of glycolysis (ECAR) to enable the cells to meet their energy demands; on the other hand, the injection of FCCP results in higher OCR due to depolarization of the mitochondrial membrane. Essentially, this assay depicts basal metabolic respiration, and following the dual injections, pushes, or stresses, the metabolic response. Based on these parameters, the software then plots OCR and ECAR of the cells by classifying the cells as aerobic, quiescent, glycolytic, or energetic state over time25,26.
ATP real-time production rate assay: This measures the cellular ATP production simultaneously from glycolysis and mitochondrial respiration. This assay quantitatively measures the metabolic shifts from the two energy pathways and provides data on the mitochondrial and glycolytic ATP production rates over time. The assay obtains basal OCR and ECAR data followed by calculating mitochondrial ATP production rate through injection of oligomycin A and glycolytic ATP production rate through injection of rotenone + antimycin A mixture (total inhibition of mitochondrial function), resulting in mitochondrial acidification17,27.
Cell mitochondria stress test (or cell mito stress test): This measures the mitochondrial function through ATP-linked respiration, quantifies cellular bioenergetics, identifies mitochondrial dysfunction, and measures cells&#39; response to stress. Various parameters, including basal and spare respiratory capacity, ATP-linked respiration, maximal respiration, and non-mitochondrial oxygen consumption, can be obtained in one assay. This assay involves sequential injections of oligomycin A, FCCP (mitochondrial uncoupling agent), a mixture of rotenone/antimycin A inhibitors to efficiently analyze the effect of these on the mitochondrial function28.
Flexibility mito fuel flex test: This measures the mitochondrial respiration rate by the oxidation of the three primary mitochondrial fuels by the presence and absence of their inhibitors. The sequential inhibition of glucose, glutamine, and fatty acids aids in measuring the dependency, capacity, and flexibility of cells and the dependency of the cells in various cellular pathways to meet the energy demand. When the mitochondria cannot meet the demands of the blocked pathway of interest by oxidizing other fuels, the cells enter a dependency state. The capacity of the cells is calculated by inhibition of the other two alternative pathways followed by the inhibition of the pathway of interest. The flexibility of cells helps in understanding the ability of mitochondria to compensate and meet the fuel needs of the inhibited pathway. It is calculated by subtracting the dependency of cells from the capacity of cells. Three different inhibitors are used independently or as a mixture of two to effectively calculate the assay parameters. 2-cyano-3-(1-phenyl-1H-indol-3-yl)-2-propenoic acid (UK5099) inhibits the oxidation of glucose by blocking the pyruvate carrier in glycolysis. Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) (BPTES) ethyl sulfide inhibits the glutamine oxidation pathway, and etomoxir inhibits the oxidation of long-chain fatty acids29.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63142/63142fig01.jpg" /> 
Figure 1: Schematic representation of the methodology for culturing and preparing osteoblasts for analysis. Murine BMSCs are isolated from long bones, cultured, and seeded in 96-well plates at 25,000 cells/well density. Culturing these cells in Osteoblast specific media is started when they reach&#160;80%-100% confluency to start their differentiation.&#160;The assays are performed at different stages of differentiation. The cartridge plates are hydrated one day prior to the assay. On the day of assay, different inhibitors are injected in the ports of the sensor cartridges based on the assay requirements, and a calibration buffer is added to the 96-well calibration plate. After calibration, the real-time cell metabolic flux assay is performed, followed by imaging the cell culture microplate using the microplate imager to normalize real-time cell metabolic flux analyzer data with cell count. <a href="https://www.jove.com/files/ftp_upload/63142/63142fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>2-cyano-3-(1-phenyl-1H-indol-3-yl)-2-propenoic acid</td>
			<td>Sigma - Aldrich</td>
			<td>PZ0160</td>
			<td>UK5099</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma - Aldrich</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide</td>
			<td>Sigma - Aldrich</td>
			<td>SML0601</td>
			<td>BPTES</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td>
			<td>Sigma - Aldrich</td>
			<td>C2920</td>
			<td>FCCP</td>
		</tr>
		<tr>
			<td>Cytation 5 imaging reader</td>
			<td>BioTek</td>
			<td>N/A</td>
			<td>Microplate imager</td>
		</tr>
		<tr>
			<td>Etomoxir sodium salt hydrate</td>
			<td>Sigma - Aldrich</td>
			<td>E1905</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342 Solution (20 mM)</td>
			<td>Thermo Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma - Aldrich</td>
			<td>I6634</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic Acid-Albumin from bovine serum</td>
			<td>Sigma - Aldrich</td>
			<td>O3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin A - 5 mg</td>
			<td>Sigma - Aldrich</td>
			<td>75351</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma - Aldrich</td>
			<td>R8875-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF 1.0 M Glucose Solution</td>
			<td>Agilent Technologies</td>
			<td>103577-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF 100mM Pyruvate Solution</td>
			<td>Agilent Technologies</td>
			<td>103578-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF 200mM Glutamine solution</td>
			<td>Agilent Technologies</td>
			<td>103579-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF DMEM media</td>
			<td>Agilent Technologies</td>
			<td>103575-100</td>
			<td>DMEM assay media eith 5mM HEPES, pH 7.4, without phenol red, sodium bicarbonate, glucose, pyruvate, and L-glutamine</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Agilent Technologies</td>
			<td>S7800B</td>
			<td>Real- Time Metabolic flux analyzer</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 FluxPak</td>
			<td>Agilent Technologies</td>
			<td>102416-100</td>
			<td>Includes XFe96 Sensor cartridges, Cell culture microplates, and Seahorse XF Calibrant solution</td>
		</tr>
		<tr>
			<td>The Cell imaging 1.1.0.11 software</td>
			<td>Agilent Technologies - BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wave software 2.6.1</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerol phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422-50G</td>
			<td></td>
		</tr>
	</tbody>
</table>

using real-time cell metabolic flux analyzer to monitor osteoblast bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63142/63142fig06.jpg" /> 
Figure 6: Representative graphs for routinely performed assays to understand the cellular bioenergetic profile of control vs. treatment group with their respective standard errors. (A) The cell energy phenotype test. The plot represents the glycolysis (ECAR) vs. mitochondrial respiration (OCR) of the control vs. two trea...

Watch this Scientific Journal Video about Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics at JoVE.com

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...

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